Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou 510080, China.
Parasitol Res. 2011 Aug;109(2):369-78. doi: 10.1007/s00436-011-2264-0. Epub 2011 Feb 23.
Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43-53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood-brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite-host interactions.
半胱氨酸蛋白酶是水解酶的超家族，在寄生虫中有许多功能。在这里，我们报道了从广州管圆线虫第四期幼虫 cDNA 文库中克隆和鉴定一种组织蛋白酶 B（AcCPB）的 cDNA。推导的氨基酸序列分析表明，AcCPB 与其他组织蛋白酶 B 家族成员具有整体保守的结构，与其他寄生虫线虫的组织蛋白酶 B 比宿主的组织蛋白酶 B 更接近，与其他生物体的同源物具有 43-53%的相似性。实时定量 PCR 分析显示，AcCPB 在第四期幼虫（L4）和第五期幼虫（L5）中的表达明显高于第三期幼虫（L3）和成虫（Aw）。出乎意料的是，AcCPB 在源自小鼠的 L4 和 L5 中的表达水平高于源自大鼠的同一阶段的幼虫。在大肠杆菌中表达的重组 AcCPB（rAcCPB）的蛋白酶活性显示出高热稳定性和酸性 pH 最佳值。来自广州管圆线虫的半胱氨酸蛋白酶抑制剂可明显抑制 rAcCPB 对卵清蛋白的消化和酶活性。此外，我们发现 rAcCPB 增加了 LPS 刺激的树突状细胞（DC）上 CD40、MHC II 和 CD80 的表达水平。在这项研究中，我们首次提供了 AcCPB 在广州管圆线虫中表达的实验证据。除了在蠕虫引起宿主血脑屏障功能障碍的 L4 和 L5 阶段高度特异性表达外，AcCPB 在非允许宿主和允许宿主来源的幼虫阶段表现出不同的表达谱，并参与 DC 的成熟，表明其在中枢神经系统入侵和寄生虫-宿主相互作用中的免疫调节中具有潜在作用。
